The present invention relates to a method for producing hydroxycarboxylic acid monomers (mostly optically active in (R)-(xe2x88x92)-configuration) by auto-degradation of polyhydroxyalkanoates (PHAs). In particular, the present invention relates to a method for producing hydroxycarboxylic acid monomers comprising the steps of: (a) synthesizing and accumulating PHAs by culturing various microorganisms; and (b) preparing optically active (R)-(xe2x88x92)-hydroxycarboxylic acids, which are monomers of PHAs, by auto-degradation of PHAs by putting the cells containing PHAs in a degradation solution such as water, salt solution, mixture of water and organic solvents, and buffer solution. The method of the present invention also includes further the separation process for the separation of the prepared (R)-(xe2x88x92)-hydroxycarboxylic acids, if they exist as a mixture of two or more (R)-(xe2x88x92)-hydroxycarboxylic acids, using liquid chromatography (LC) or high performance liquid chromatography (HPLC), and also includes further the purification process for the removal of impurities from the purely-separated (R)-(xe2x88x92)-hydroxycarboxylic acids by organic solvent extraction and the powder-making process of the purified (R)-(xe2x88x92)-hydroxycarboxylic acids.
(R)-(xe2x88x92)-3-hydroxycarboxylic acid can be widely used as a chiral precursor for several reasons as follows: it contains two functional groups, hydroxyl-group and carboxyl-group; the functional groups are convenient to modify; and a new chiral center can be introduced. And (R)-(xe2x88x92)-3-hydroxycarboxylic acid can be widely used as a precursor for synthesizing antibiotics, vitamins, aromatics, pheromones and the like; as a material for developing non-peptide ligand used in drug design; as a precursor for developing new drug; and especially as a precursor of carbapenem antibiotics, which has recently been drawing much attention to replace xcex2-lactam antibiotics such as penicillin (Scott, In: Asymmetric Synthesis, Morrison and Scott, Eds., Academic Press Inc., Orlando, Fla., 1984). In addition, it was reported that (+)-thienamycin could be synthesized from methyl-(R)-(xe2x88x92)-3-hydroxybutyrate (Chiba and Nakai, Chem,. Lett., 651-654, 1985; Seebach and Zuger, Helvetica Chim. Acta, 65: 495-503, 1982)
Presently, (R)-(xe2x88x92)-3-hydroxycarboxylic acid is mainly produced by the following methods: oxidation of aliphatic glycol by fermentation process (Harada and Hirayashi, Agric. Biol. Chem., 32: 1175, 1968); (R)-(xe2x88x92)-xcex2-hydroxylation of carboxylic acid using microorganisms (Hasegawa et al., J. Ferment. Technol., 60: 501, 1982); and hydrogenation of xcex2-diketone using chiral catalyst (Noyori et al., J. Am. Chrem. Soc., 109: 5856, 1987; Brussel et al., WO97/14500A1, 1997).
Polyhydroxyalkanoates (PHAs) are a carbon and/or energy storage material synthesized and accumulated in numerous microorganisms (Anderson and Dawes, Microbiol. Rev., 54: 450-472, 1990). More than 120 kinds of monomers have been found to be the constituents of PHAs, which can vary depending on the cultured microorganisms, chemical substrate or cosubstrate used, and culture conditions (Lee, Biotechnol. Bioeng., 49: 1-14, 1996; Steinbuchel and Valentin, FEMS Microbiol. Lett., 128: 219-228, 1995). Optically-pure (R)-(xe2x88x92)-hydroxycarboxylic acids may be easily prepared by degrading biosynthesized PHAs since the monomer units of biosynthesized PHAs are composed of monomers all in (R)-(xe2x88x92)-configuration, if the monomer has chiral center on the carbon possessing hydroxyl group, due to the optical specificity of biosynthetic enzymes. A method for producing (R)-(xe2x88x92)-3-hydroxyburyric acid and (R)-(xe2x88x92)-3-hydroxyvaleric acid from poly-(R)-(xe2x88x92)-3-hydroxybutyrate (PHB) or poly-(R)-(xe2x88x92)-3-hydroxybutyrate-co-(R)-(xe2x88x92)-3-hydroxyvalerate (PHB/V) by chemical degradation was reported (Seebach et al., Org. Synth., 71: 39-47, 1992; Seebach and Zuger, Helvetica Chim. Acta, 65: 495-503, 1982; Pennetreau, US005107016A, 1992; Pennetreau, EP0377260A1, 1989).
However, in the above method of producing (R)-(xe2x88x92)-3-hydroxybutyric acid and (R)-(xe2x88x92)-3-hydroxyvaleric acid by chemical degradation, organic solvents were used in large amounts, and the production efficiency was rather low due to complicated processes. Therefore, a new method for producing optically active hydroxycarboxylic acid, which can solve the above problem, is highly required in this field.
Very recently, a method for producing (R)-(xe2x88x92)-3-hydroxybutyric acid by microorganisms has been reported (Akira and Tatsuhiko, JP9-234091, 1997). This method was based on the simple obvious assumption that some microorganisms which accumulate poly-(R)-(xe2x88x92)-3-hydroxybutyrate (PHB) would also be able to produce its monomer, (R)-(xe2x88x92)-3-hydroxybutyric acid. They screened for micrcorgarisms that produce (R)-(xe2x88x92)-3-hydroxybutyric acid, and found that Pseudomonas sp., Burkholderia sp. and Alcaligenes eutrophus were able to produce (R)-(xe2x88x92)-3-hydroxybutyric acid. Here, it should be noted that Alcaligenes eutrophus has recently been renamed as Ralstonia eutropha, and therefore, does not belong to the family of Alcaligenes anymore (Yabuuchi et al., Microbiol. Immunol., 39: 897-904, 1995). In the above method, microorganisms were cultivated for several days (4-7 days), and then transferred to potassium phosphate buffer solution for the production of (R)-(xe2x88x92)-3-hydroxybutyric acid. The monomer yields were very low at 2-8%. Furthermore, production of only (R)-(xe2x88x92)-3-hydroxybutyric acid, but not other (R)-(xe2x88x92)-hydroxycarboxylic acids, was reported. Since the monomer yields were extremely low and it took several days resulting in low productivity (defined as gram product produced per unit volume per unit time), the above method is not suitable for industrial applications.
PHAs are synthesized and accumulated inside the cells usually when one of the growth factors, such as nitrogen, phosphorus, oxygen, potassium and sulfur, is limiting while carbon source is in excess (Anderson and Dawes, Microbiol. Rev., 54: 450-472, 1990). Thus, if the limiting growth factor is supplied again, cells degrade the accumulated PHAs and grow.
All microorganisms that synthesize PHAs contain intracellular PHA depolymerase as well as PHA biosynthesis enzymes. Intracellular PHA depolymerase is known to exist in two states, soluble form and attached form to PHA granules (Merrick and Dourdoroff, J. Bacteriol., 88: 60-71, 1964; Merrick et al., J. Bacteriol., 89: 234-239, 1965; Merrick and Yu, Biochem., 5: 3563-3568, 1966; Griebel et al., Biochemn., 7: 3676-3681, 1968; Griebel and Merrick, J. Bacteriol., 108: 782-789, 1971; Anderson and Dawes, Microbiol. Rev., 54: 450-472, 1990). Merrick and Doudoroff (J. Bacteriol. 88: 60-71, 1964) showed that native poly-(R)-(xe2x88x92)-3-hydroxybutyrate (PHB) granules, but not the solvent extracted PHB granules, could be depolymerized by an intracellular depolymerase system. They demonstrated that the PHB granules accumulated in Bacillus megaterium could be hydrolyzed to (R)-(xe2x88x92)-3-hydroxybutyric acid by a crude enzyme fraction of PHB-depleted cells of Rhodospirillum rubrum. Furthermore, Hippe and Schlegel (Arch. Mikrobiol. 56: 278-299, 1967) reported that the soluble depolymerase from Alcaligenes (Hydrogenomonas) spp. could degrade native PHB to give (R)-(xe2x88x92)-3-hydroxybutyric acid. These early studies clearly demonstrated that cells possess intracellular PHA depolymerases, and can degrade PHA into monomers. However, isolation of native (amorphous) PHA granules is not only complicated but also expensive in large scale. Furthermore, isolation of intracellular depolymerase for the depolymerization of PHAs is also cumbersome and expensive. If crude cell extract containing intracellular PHA depolymerase is used for degradation of PHAs into monomers, there is a significant problem of product purification due to the contamination by the components in the crude cell extracts. Another problem that hinders the production of monomers by degradation of PHAs by employing cells is that the degraded products, the monomers of PHAs, are further metabolized by the cells. Therefore, it is imoossible to produce monomers by degradation of PHAs unless the metabolic pathways that further metabolize hydroxycarboxylic acid monomers are made to be inactive or blocked. The present invention is based on the assumption that it may be possible to make the metabolic pathways degrading the hydroxycarboxylic acid monomers inactive while maintaining the activity of the intracellular PHA depolymerases by controlling the environmental factors. This idea is clearly different from the method of Akira and Tatsuhiko (JP9-234091, 1997) in that the intracellular PHA depolymerase system is to be utilized for the production of (R)-(xe2x88x92)-hydroxycarboxylic acid monomers in the present invention, while Akira and Tatsuhiko""s method is based on the ability of 3 newly isolated microorganisms (Pseudomonas sp., Burkholderia sp. Rlastonia eutropha (formerly known as Alcaligenes eutrophus; Yabuuchi et al., Microbiol. Immunol., 39: 897-904, 1995)) that are able to produce (R)-(xe2x88x92)-3-hydroxybutyric acid. And, therefore, the present invention begins from the assumption that the synthesized PHAs may be auto-degraded from the intact cells and the monomers can be released into the extracellular environment by controlling the environmental conditions. Therefore, the present invention provides methods to produce various optically active (R)-(xe2x88x92)-hydroxycarboxylic acid monomers efficiently and economically with high yields.
The present inventors have successfully developed a new method for producing optically active hydroxycarboxylic acid monomers from PHAs accumulated in microorganism. The method of the present invention comprises the steps of: (a) synthesizing and accumulating PHAs by culturing various microorganisms; and (b) preparing hydroxycarboxylic acids, which are monomers of PHAs (mostly optically active in (R)-(xe2x88x92)-configuration) by auto-degradation of PHAs by keeping the cultured microorganism in a degradation solution such as water, salt solution, mixture of water and organic solvents, and buffer solution. The present method involves separation process of the prepared (R)-(xe2x88x92)-hydroxycarboxylic acids, using liquid chromatography (LC) or high performance liquid chromatography (HPLC), and also involves purification process of removing impurities from the purely-separated (R)-(xe2x88x92)-hydroxycarboxylic acids by organic solvent extraction and powder-making process of drying the purified (R)-(xe2x88x92)-hydroxycarboxylic acids.
Finally, the object of the present invention is to provide a new method for producing various optically active (R)-(xe2x88x92)-hydroxycarboxylic acid monomers with high yields comprising the steps of: (a) synthesizing and accumulating PHAs by culturing various microorganisms, including recombinant strains which can accumulate and intracellularly degrade PHAs; and (b) preparing optically active (R)-(xe2x88x92)-hydroxycarboxylic acid monomers by auto-degradation of PHAs.
The object of the present Invention is also to provide a method for producing (R)-(xe2x88x92)-hydroxycarboxylic acid monomers further comprising the steps of separating the prepared (R)-(xe2x88x92)-hydroxycarboxylic acid monomers, using liquid chromatography (LC) or high performance liquid chromatography (HPLC).
The object of the present invention is also to provide a method for producing final (R)-(xe2x88x92)-hydroxycarboxylic acids powder product further comprising the steps of purifying the purely-separated (R)-(xe2x88x92)-hydroxycarboxylic acids by organic solvent extraction; and making powders of the purified (R)-(xe2x88x92)-hydroxycarboxylic acids by drying.